Paternal uniparental isodisomy of chromosome 20q--and the resulting changes in GNAS1 methylation--as a plausible cause of pseudohypoparathyroidism

Abstract

Heterozygous inactivating mutations in the GNAS1 exons (20q13.3) that encode the alpha-subunit of the stimulatory G protein (Gsalpha) are found in patients with pseudohypoparathyroidism type Ia (PHP-Ia) and in patients with pseudo-pseudohypoparathyroidism (pPHP). However, because of paternal imprinting, resistance to parathyroid hormone (PTH)-and, sometimes, to other hormones that require Gsalpha signaling-develops only if the defect is inherited from a female carrier of the disease gene. An identical mode of inheritance is observed in kindreds with pseudohypoparathyroidism type Ib (PHP-Ib), which is most likely caused by mutations in regulatory regions of the maternal GNAS1 gene that are predicted to interfere with the parent-specific methylation of this gene. We report a patient with PTH-resistant hypocalcemia and hyperphosphatemia but without evidence for Albright hereditary osteodystrophy who has paternal uniparental isodisomy of chromosome 20q and lacks the maternal-specific methylation pattern within GNAS1. Since studies in the patient's fibroblasts did not reveal any evidence of impaired Gsalpha protein or activity, it appears that the loss of the maternal GNAS1 gene and the resulting epigenetic changes alone can lead to PTH resistance in the proximal renal tubules and thus lead to impaired regulation of mineral-ion homeostasis.

Figure 1

A, Haplotype analysis of genomic DNA from K-1 and his immediate family (K-2 = sister; K-3 = mother; K-4 = father), across chromosome 20. The fully informative markers on 20q reveal LOH for K-1, with a lack of maternal contribution. B, Schematic presentation of GNAS1 gene (top) and allele-specific methylation of GNAS1, in K-1 and his relatives (bottom). Exons encoding sense transcripts are depicted by white boxes; those encoding antisense transcripts are depicted by gray boxes. Thicker lines indicate probes used in Southern analysis: NESP55 (nucleotides 317–1705; GenBank accession number AJ009849), XLαs (nucleotides 80–1693; GenBank accession number AJ224868), AS (nucleotides 11646–13156; GenBank accession number AJ251760), and A/B (nucleotides 28580–31035; GenBank accession number AL121917). Normal methylation status of the parental alleles is marked by a plus sign (+) or a minus sign (−). Exons 1–3, encoding portions of Gsα, are depicted by black boxes; exons 4–13 are not included. Combinations of restriction enzymes—Bg = BglII; P = PvuI; E = EcoRV; F = FspI; S = SacI; N = NotI; B = BamHI; Nr = NruI (note that the small internal fragments of the EcoRV/FspI digest were not transferred)—are indicated below autoradiographies, and the sizes (in kb) of the expected DNA fragments are indicated.

Figure 2

A, Basal cAMP accumulation (white bars), and accumulation of this second messenger after challenge with 1 μM of either PTH(1-34) (gray bars) or isoproterenol (black bars), in fibroblasts derived from K-1 and his parents, K-3 and K-4. The data represent the mean ±SEM of three independent experiments. B, Assessment of Gsα-protein levels by western blot analysis. Equal amounts of protein were loaded in each lane and were separated by 9% SDS-PAGE. After transfer to nitrocellulose, detection of Gsα protein was achieved with an anti-Gsα antibody and a horseradish peroxidase–labeled second antibody. The positions of protein size markers (in kD) are indicated. The findings are representative of four independent experiments with similar results.

Figure 3

Genetic mechanisms that are predicted to underlie the different forms of PHP. In healthy individuals (normal), the paternal allele (P) gives rise to sense and antisense transcripts (solid-shaft arrows) that are derived from the nonmethylated promoter regions upstream of exons XL, AS, and A/B, whereas the transcript that encodes NESP55 is derived from the maternal allele (M). Note that Gsα transcripts are derived in proximal renal tubules and possibly in other tissues from the maternal allele and not from the paternal allele (dotted-shaft arrows). In patients with PHP-Ia, mutations within 1 of the 13 Gsα-specific exons on the maternal allele (thick X) lead to either a complete lack of Gsα protein or a functionally impaired protein. In contrast, patients with PHP-Ib are presumed to carry a mutation in regulatory regions of the maternal GNAS1 allele (light X, over “Gαs”) that causes loss of imprinting at exon A/B and that leads to impaired Gsα expression in some tissues (tripled X). Lack of a maternal GNAS1 allele—as in K-1, who has patUPD20q—is predicted to result in a lack of Gsα transcripts in the renal cortex that leads to PTH-resistant hypocalcemia and hyperphosphatemia.